A Phase 0 Trial of Hydroxychloroquine in Patients with Stage III and IV Resectabl

羟氯喹治疗 III 期和 IV 期可切除患者的 0 期试验

• 批准号: 7892776
• 负责人: Janice M. Mehnert
• 金额: $ 32.02万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-07 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892776
• 关键词: Address; Adverse effects; Affect; Aftercare; Allografting; Animal Model; Antineoplastic Agents; Ants; Apoptosis; Apoptotic; Autophagocytosis; Autophagosome; Binding Proteins; Biological Assay; Biopsy; Biopsy Specimen; Blood; Catabolic Process; Cell Death; Cell Line; Cell Survival; Cells; Chloroquine; Cleaved cell; Clinical; Clinical Trials; Clinical Trials Design; Connective Tissue Diseases; Correlative Study; Detection; Development; Digestion; Dose; Electron Microscopy; Evaluation; Excision; FDA approved; Future; Glucose; Goals; Gold; Homeostasis; Human; Hydroxychloroquine; Imagery; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro; Individual; Knowledge; Laboratories; Lysosomes; Malaria; Malignant Neoplasms; Measures; Mediating; Melanoma Cell; Membrane; Metabolic stress; Methods; Mitotic; Modeling; Molecular Chaperones; Mus; Necrosis; Organelles; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Polyubiquitin; Process; Proteins; Recycling; Sampling; Skin; Skin Tissue; Specimen; Staging; Staining method; Stains; Stimulus; Stress; Sum; Surrogate Markers; Techniques; Testing; Time; Tissue Sample; Tumor Tissue; Vesicle; Western Blotting; cancer cell; cancer therapy; caspase-3; cytotoxic; drug efficacy; glucose-regulated protein 170; human tissue; in vivo; inhibition of autophagy; inhibitor/antagonist; melanocyte; melanoma; molecular marker; neoplastic cell; new therapeutic target; novel; palliative; programs; protein degradation; public health relevance; research study; response; standard measure; stress tolerance; therapeutic development; treatment effect; tumor

DESCRIPTION (provided by applicant): Autophagy enables tumors to survive metabolic stress in a dormant state for prolonged periods of time and as such, this pathway is an emerging target for the creation of new anticancer therapies. Hydroxychloroquine (HCQ) is a drug that blocks autophagy, raising intralysosomal pH and inhibiting the final step of this pathway, impairing autophagic protein degradation. This results in autophagosome accumulation, potentially disturbing the survival of tumor cells reliant on autophagy and shunting them toward apoptotic and/or necrotic cell death. Preliminary results in melanoma cell lines indicate that treatment with chloroquine (CQ), a similar drug to HCQ used readily in the laboratory, increases autophagosome accumulation, and increased cell death was noted from treatment with CQ at varying concentrations. Additionally, CQ induced autophagosome accumulation in a mammalian tumor allograft model. Given that the autophagy pathway may be active in melanoma, we hypothesize that administration of HCQ can modulate the process of autophagy in melanoma tumor tissue samples in humans in vivo. To test this hypothesis, a phase 0 trial of HCQ in patients with stage III or IV melanoma planned for palliative or curative resection is proposed. Specifically, we will look for signs of autophagy inhibition by HCQ, comparing the mean number of autophagic vesicles per cell seen on electron microscopy (EM) in pre- versus post- treatment tumor specimens. Pre- and post- treatment skin samples and PBMCs will similarly be assayed to explore the potential of these samples as surrogate markers for treatment effect. While autophagosome accumulation on EM is a reliable method for detection of autophagy inhibition, it is expensive, tedious and requires a skilled individual to perform and hence, is not widely applicable to clinical specimens to detect autophagy inhibition. Thus, as part of this trial we will examine several proteins shown to detect inhibition of autophagy in laboratory models and in some preliminary studies of human tumor tissue, with the hope that these simpler assays will facilitate autophagy detection in human samples. Markers that will be examined include Beclin1, an essential autophagy regulator, and LC3, which is cleaved into LC3-I and LC3- II, with the latter translocated to the autophagosome membrane during autophagy. In addition, our group has discovered that autophagy deficient tumors under stress accumulate p62, a polyubiquitin binding protein that delivers aggregates to the lysosome for degradation, as well as several chaperone proteins including glucose related protein 70 (GRp170). It is expected that treatment with HCQ in melanomas with functional autophagy will result in increased autophagosome formation on EM and increased levels of LC3-II, p62 and GRp170 by immunohistochemistry and Western blot. Potential proapoptotic and/or ant proliferative effects of HCQ will also be explored through the use of pre- and post-treatment activated caspase-3, TUNEL, Ki-67 and mitotic rate assays. The goal of this proposal is to modulate autophagy for the first time in human tumors, using results from these experiments to guide development of markers of autophagy in future clinical trials. PUBLIC HEALTH RELEVANCE: We propose a preliminary trial in humans with melanoma (a Phase O trial) to see if administering hydroxychloroquine (HCQ), a FDA approved drug with little to no side effects, can block the process of autophagy, a survival mode for several different types of tumor cells. We will use a method called electron microscopy to study if HCQ is inhibiting autophagy in melanoma tumor tissue and blood and skin samples, but since this method is expensive and laborious we will also examine four proteins -- Beclin1, LC3, p62 and GRp170 -- which detect autophagy inhibition in cell lines, animal models, and some preserved human tissue samples to demonstrate that these simpler tests may also detect inhibition of autophagy in human beings. Our hope is that we will be able to use these results to better understand and detect autophagy, making it easier to develop future drugs against this pathway in cancer.

描述（由申请人提供）：自噬使肿瘤能够在长时间的休眠状态下存活代谢应激，因此，这一途径是创造新的抗癌疗法的新兴靶点。羟氯喹（Hydroxychloroquine， HCQ）是一种阻断自噬的药物，可提高溶酶体内pH值，抑制自噬途径的最后一步，损害自噬蛋白降解。这导致自噬体积累，潜在地干扰依赖自噬的肿瘤细胞的存活，并使其转向凋亡和/或坏死细胞死亡。黑色素瘤细胞系的初步结果表明，氯喹（CQ）治疗增加了自噬体的积累，并且不同浓度的氯喹治疗增加了细胞死亡。氯喹与实验室中常用的HCQ类似。此外，CQ在哺乳动物肿瘤同种异体移植模型中诱导自噬体积累。鉴于自噬途径在黑色素瘤中可能是活跃的，我们假设给药HCQ可以调节人体内黑色素瘤肿瘤组织样本的自噬过程。为了验证这一假设，提出了一项HCQ在III期或IV期黑色素瘤患者中进行姑息性或治愈性切除的0期试验。具体来说，我们将通过HCQ寻找自噬抑制的迹象，比较治疗前和治疗后肿瘤标本中每个细胞在电子显微镜（EM）上看到的自噬囊泡的平均数量。治疗前和治疗后的皮肤样本和pbmc也将进行类似的分析，以探索这些样本作为治疗效果替代标记物的潜力。虽然自噬体在电镜上的积累是检测自噬抑制的一种可靠的方法，但它昂贵、繁琐，需要熟练的个人来完成，因此不广泛适用于临床标本检测自噬抑制。因此，作为本试验的一部分，我们将在实验室模型和一些人类肿瘤组织的初步研究中检查几种显示可检测自噬抑制的蛋白质，希望这些更简单的分析将有助于在人类样本中检测自噬。我们将研究的标志物包括Beclin1（一种重要的自噬调节因子）和LC3（在自噬过程中被裂解为LC3- i和LC3- II，后者在自噬过程中易位到自噬体膜上）。此外，我们的研究小组发现，在应激条件下，自噬缺陷的肿瘤会积累p62（一种多聚素结合蛋白，可将聚集体传递给溶酶体降解），以及几种伴侣蛋白，包括葡萄糖相关蛋白70 （GRp170）。免疫组织化学和Western blot结果显示，HCQ治疗功能性自噬的黑色素瘤可导致EM上自噬体形成增加，LC3-II、p62和GRp170水平升高。HCQ的潜在促凋亡和/或蚂蚁增殖作用也将通过使用处理前和处理后的活化caspase-3、TUNEL、Ki-67和有丝分裂率测定来探索。本提案的目标是首次在人类肿瘤中调节自噬，利用这些实验的结果来指导未来临床试验中自噬标志物的开发。